1. Field of the Invention
The present invention relates to a protein having an amino acid sequence identical to or substantially identical to an amino acid sequence represented by SEQ ID NO.: 1 (preferably, a protein having the Na+xe2x80x94HCO3xe2x88x92 cotransporter activity: hereinafter, abbreviated as present protein in some cases) or a salt thereof and a DNA encoding it.
2. Description of the Related Art
The organisms maintain and regulate the intracellular ionic environment so that the cellular functions such as proliferation, differentiation, contraction and secretion can exert to a maximum extent as necessary. In order to regulate the ionic environment to the conditions optimal for existence, there are a plurality of transporter proteins transporting ions such as Na+, Ca2+, H+ and HCO3xe2x88x92 in a plasma membrane. A Na+xe2x80x94HCO3xe2x88x92 cotransporter belonging to such the transporter is driven by the concentration gradient of Na+ inside and outside a cellular membrane, and take one Na+ into a cell together with one or more HCO3xe2x88x92 ions [Cellular Molecular Biology, 3rd edition, translated under supervision by Keiko Nakamura et al., Kyoikusha]. Since the structure of a protein such as an enzyme which functions in cells is greatly affected by pH, there is an optimal pH for the protein function. For that reason, maintenance and regulation of an intracellular pH is extremely important to cells for maintaining homeostasis of the cellular function. Since a Na+xe2x80x94HCO3xe2x88x92 cotransporter exists in the cell membrane and HCO3xe2x88x92 taken into cells by a Na+xe2x80x94HCO3xe2x88x92 cotransporter neutralizes H+ in a cytoplasmic sol, it plays an important role in regulating an intracellular pH like a Na+xe2x80x94H+ exchanger [Cellular Molecular Biology, 3rd edition, translated under supervision by Keiko Nakamura et al., Kyoikusha].
In addition, the Na+xe2x80x94HCO3xe2x88x92 cotransporter is present in a proximal convoluted uriniferous tubule of a kidney and exerts in resorting HCO3xe2x88x92 from a glomerulus filtrate in cooperation with a Na+xe2x80x94H+ exchanger [The Journal of Biological Chemistry, 272, 19111 (1997)].
The Na+xe2x80x94HCO3xe2x88x92 cotransporter plays an important role in maintaining the cell homeostasis at a normal time, and is involved in occurrence of cell disorder at an ischemic time. Since intracellular acidosis is caused by ischemia and a Na+xe2x80x94HCO3xe2x80x94 cotransporter and a Na+xe2x80x94H+ exchanger function in order to regulate the decrease in pH. As a result, the intracellular Na+ concentration is elevated and the overload of Ca2+ into cells is caused via a Na+xe2x80x94Ca2+ exchanger. Consequently, this overloaded Ca2+ is considered to be one of factors causing cell disorder [Basic Research in Cardiology, 91, 191 (1996)].
The kidney-derived Na+xe2x80x94HCO3xe2x88x92 cotransporter gene has been cloned from an amphibian [Nature, 387, 409(1997)]], a rat [American Journal of Physiology, 274, F425(1998)], and a human being [The Journal of Biological Chemistry, 272, 19111, (1997)]. However, a Na+xe2x80x94HCO3xe2x88x92 cotransporter gene which is expressed in mainly organs other than kidney such as heart and brain has not hitherto been known.
The object of the present invention is to provide a protein having an amino acid sequence identical to or substantially identical to an amino acid sequence represented by SEQ ID NO.: 1 (preferably, a protein having the Na+xe2x80x94HCO3xe2x88x92 cotransporter activity), a partial peptide thereof, or a salt thereof, a DNA encoding a protein having an amino acid sequence identical to or substantially identical to an amino acid sequence represented by the SEQ ID NO.: 1 (preferably, a protein having the Na+xe2x80x94HCO3xe2x88x92 cotransporter activity), or a partial peptide thereof, a recombinant vector containing the DNA, a transformant harboring the recombinant vector a process for producing a protein having an amino acid sequence identical to or substantially identical to an amino acid sequence represented by the SEQ ID NO.: 1 (preferably, a protein having the Na+xe2x80x94HCO3xe2x88x92 cotransporter activity), or a salt thereof, an antibody to a protein having an amino acid sequence identical to or substantially identical to an amino acid sequence represented by the SEQ ID NO.: 1 (preferably, a protein having the Na+xe2x80x94HCO3xe2x88x92 cotransporter activity), a partial peptide thereof, or a salt thereof, a method for screening a compound which alters the Na+xe2x80x94HCO3xe2x88x92 cotransporter activity of a protein having an amino acid sequence identical to or substantially identical to an amino acid sequence represented by the SEQ ID NO.: 1 (preferably, a protein having the Na+xe2x80x94HCO3xe2x88x92 cotransporter activity), or a salt thereof, a compound obtained by the screening method, or a salt thereof, and a pharmaceutical composition containing the compound or a salt thereof.
The present inventors studied extensively and, as a result, we isolated a cDNA encoding a Na+xe2x80x94HCO3xe2x88x92 cotransporter protein specifically expressed in a human-derived heart, analyzed the whole nucleotide sequence and, thereafter, succeeded in expression of it in a cell. And, based on these findings, the present inventors further studied, which resulted in completion of the present invention.
That is, the present invention relates to:
(1) a protein having an amino acid sequence identical to or substantially identical to an amino acid sequence represented by SEQ ID NO.: 1, or a salt thereof,
(2) a protein described in (1) above having a Na+xe2x80x94HCO3xe2x88x92 cotransporter activity, or a salt thereof,
(3) a partial peptide of the protein described in (1) above, or an amide thereof, or an ester thereof, or a salt thereof,
(4) a DNA containing the DNA having a nucleotide sequence encoding the protein described in (1) above,
(5) the DNA described in (4) above having a nucleotide sequence represented by SEQ ID NO.: 2,
(6) a recombinant vector containing the DNA described in (5) above,
(7) a transformant transformed with the recombinant vector described in (6) above,
(8) a method for producing the protein described in (1) above, or a salt thereof, which comprises culturing the transformant described in above (7), to produce the protein described in (1) above which is accumulated and taken,
(9) an antibody against the protein described in (1) above, or a salt thereof, or the partial peptide described in (3) above, or an amide thereof, or an ester thereof, or a salt thereof,
(10) a method for screening a compound, or a salt thereof, which promotes or inhibits a Na+xe2x80x94HCO3xe2x88x92 cotransporter activity of the protein described in (1) above, or a salt thereof, or the partial peptide described in (3) above, or an amide thereof, or an ester thereof, or a salt thereof, which comprises using the protein described in (1) above, or a salt thereof, or the partial peptide described in (3) above, or an amide thereof, or an ester thereof, or a salt thereof,
(11) a kit for screening a compound, or a salt thereof, which promotes or inhibits a Na+xe2x80x94HCO3xe2x88x92 cotransporter activity of the protein described in (1) above, or a salt thereof, or the partial peptide described in (3) above, or an amide thereof, or an ester thereof, or a salt thereof, which comprises the protein described in (1) above, or a salt thereof, or the partial peptide thereof described in (3) above, or an amide thereof, or an ester thereof, or a salt thereof,
(12) a compound or a salt thereof which promotes or inhibits a Na+xe2x80x94HCO3xe2x88x92 cotransporter activity of the protein described in (1) above, or a salt thereof, or the partial peptide described in claim 3, or an amide thereof, or an ester thereof, or a salt thereof, which is obtainable by using the screening method described in (10) above or the kit for screening described in (11) above,
(13) a pharmaceutical composition which comprises a compound or a salt thereof which promotes or inhibits a Na+xe2x80x94HCO3xe2x88x92 cotransporter activity of the protein described in (1) above, or a salt thereof, or the partial peptide described in (3) above, or an amide thereof, or an ester thereof, or a salt thereof, which is obtainable by using the screening method described in (10) above or the kit for screening described in (11) above,
(14) the pharmaceutical composition described in (13) above which is a composition for preventing and/or treating for cardiac infarction or dysfunction accompanying therewith, unstable angina, restenosis after PTCA, arrhythmia, heart failure, hypertension and tissue disorder accompanying therewith, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage and cerebral disorder accompanying therewith,
(15) a composition for diagnosing cardiac infarction or dysfunction accompanying therewith, unstable angina, restenosis after PTCA, arrhythmia, heart failure, hypertension and tissue disorder accompanying thereof, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage and cerebral disorder accompanying therewith, which comprises the antibody described in (9) above, and
(16) a method for quantitating the protein described in (1) above, or a salt thereof, or the partial peptide described in (3) above, or an amide thereof, or an ester thereof, or a salt thereof, which comprises using the antibody described in (9) above.
More particularly, the present invention provides:
(17) the Na+xe2x80x94HCO3xe2x88x92 transporter protein described in (1) above or a salt thereof, wherein the protein is a protein containing [1] an amino acid sequence in which 1 or 2 or more (preferably around 1 to 30, more preferably around 1 to 9, further preferably a few (1 or 2)) amino acids in an amino acid sequence represented by SEQ ID NO.: 1 are deleted, [2] an amino acid sequence in which 1 or 2 or more (preferably around 1 to 30, more preferably around 1 to 10, further preferably a few (1) or (2)) amino acids are added to an amino acid sequence represented by SEQ ID NO.: 1, [3] an amino acid sequence in which 1 or 2 or more (preferably around 1 to 30, more preferably around 1 to 10, further preferably a few (1 or 2)) amino acids in an amino acid sequence represented by SEQ ID NO.: 1 are substituted with other amino acids, or [4] an amino acid sequence comprising a combination of them,
(18) the method described in (10) above, wherein the case where the protein described in (1) above, or a salt thereof, or the partial peptide described in (3) above, or an amide thereof, or an ester thereof, or a salt thereof is contacted with a test compound and the contrary case are compared,
(19) a method for screening a compound or a salt thereof which alters a Na+xe2x80x94HCO3xe2x88x92 cotransporter activity, which comprises comparing the case where a test compound is contacted with a cell containing the protein described in (1) above and the contrary case
(20) a method for screening a compound or a salt thereof which alters a Na+xe2x80x94HCO3xe2x88x92 cotransporter activity, which comprises comparing the case where a test compound is contacted with the protein described in (1) above which is expressed on a cell membrane of the transformant described in (7) above by culturing the transformant and the contrary case
(21) a compound which alters a Na+xe2x80x94HCO3xe2x88x92 cotransporter activity of the protein described in (1) above, or a salt thereof, which is obtainable by the screening method described in (18) to (20) above, and
(22) a composition for preventing and/or treating cardiac infarction or dysfunction accompanying therewith, unstable angina, restenosis after PTCA, arrhythmia, heart failure, hypertension and tissue disorder accompanying thereof, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage and cerebral disorder accompanying therewith, which comprises a compound or a salt thereof which alters the function of the protein described in (1) above, which is obtainable by the screening method described in (18) to (20) above.